This disclosure relates to photoactivators, in particular photoactivators for use with single- and multi-photon excitation, methods of use of such photoactivators, and the articles derived therefrom.
Tissue engineering is an emerging field because of the unlimited potential to either repair existing tissue or organs or to create them anew. A major challenge in tissue engineering is reproduction of the features of tissues such as skin, muscle, and bone, which are complex, three-dimensional objects on the sub-micrometer scale. Known fabrication techniques such as photolithography and micro-contact printing have severe limitations for tissue engineering because they are not readily amenable to forming three-dimensional structures. Thus, although photolithography can produce features on the 150 nanometer (nm) scale, it is essentially a two-dimensional technique. In addition, photolithographic systems are not water-compatible. Micro-contact printing, or stamping, can produce features on the 500 nm to one micrometer (μm) level, but is also essentially a two-dimensional technique. While some proteins have been stamped, both the available morphologies (essentially two-dimensional) and the reactive chemistries, typically thiols, are quite limited.
Some methods of forming three-dimensional structures in tissue engineering are known, primarily molding-based approaches. These are applicable primarily to synthetic polymers, however, and have a number of drawbacks when applied to naturally occurring molecules such as proteins. Another technique, crosslinking, is also of limited use with naturally occurring molecules, because such molecules are not readily amenable to crosslinking using the currently available chemistries. A two-step crosslinking process for proteins has been reported, using a crosslinking agent comprising a chemically activatable site and a photoactivatable site. In this process the agent is first chemically reacted with a first protein, followed by the photochemical-mediated reaction with a second protein. In addition to the drawback of being a two-step process, like all chemical crosslinking methods this process lacks spatial control and is thus inadequate to reproduce the fine features of real tissues.
One important natural protein is collagen; in fact, the most abundant natural protein is Type 1 collagen. Collagen is a major building block for a wide range of tissues, including skin, muscle, teeth, and bone. Methods to cast or crosslink collagen are known, but the reported methods for casting collagens into three-dimensional shapes are not suitable for providing structural features on the micron and submicron size scale. Collagens are also difficult to crosslink, as known photochemical methods often utilize basic enviroments (pH greater than 7) but most collagens are soluble only in acidic environments. Techniques to assemble proteins such as collagens into three-dimensional structures on a sub-micron scale would be of considerable utility in the fabrication of artificial tissue and other tissue engineering applications. Accordingly, there remains a need in the art for techniques to facilitate crosslinking between proteins such as collagen (type 1 collagen, in particular) to create artificial three-dimensional tissue structures on a sub-micron level. There additionally remains a need for methods of free-form fabrication of two- and three-dimensional tissue structures having dimensions or features in the sub-micron range, especially techniques suitable for synthesis using biomolecular subunits such as proteins, peptides, and oligonucleotides, as well as bioactive small molecules such as hormones, cytokines and drugs.